Rift Valley Fever (RVF) is a mosquito-borne viral zoonosis caused by RVFV in humans and livestock. Currently, there are no approved vaccines or antiviral therapies available. Additionally, in Saudi Arabia, there is a lack of a routine screening system to monitor RVFV in humans and animals which hinders to design and develop the preventive measures as well as the prediction of future outbreaks and the potential re-emergence of RVFV. Hence, we have performed the cloning, sequencing, and phylogenetic analysis, of nucleocapsid (N) protein gene. The sequence analysis showed high similarities with RVFV isolates reported from humans and animals. The highest similarity (99.5%) was observed with an isolate from Saudi Arabia (KU978775-Human) followed by 99.1% with four RVFV isolates (Human and Bovine) from other locations. A total of 51 nucleotides and 31 amino acid variations were observed throughout the N protein gene sequences. The phylogenetic relationship formed closed clusters with other isolates collected from Saudi Arabia. Thus, we report of the cloning, sequencing, and phylogenetic analysis of the RVFV-N protein gene from Saudi Arabia.